數株益生菌及其發酵乳抑制大腸癌細胞增生及4NQO誘導Int-407基因毒性之研究

Inhibition of colon cancer cell proliferation and genotoxicity of 4-nitroquinoline-N-oxide on human intestine cell by several probiotics and fermented milk

朱芳瑢(Fang-jung Chu)

指導教授 ： 游若篍

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02859

摘要

結腸直腸癌為國人主要的癌症死因，約有70 % 的結腸直腸癌與飲食有關。近年來益生菌的相關研究指出，益生菌及其製品對致突變劑和致突變原具有抗致突變和抗腫瘤等功效，因此本研究以過去本實驗室篩選出之具有較佳耐酸性pH、膽汁及對 B[a]P、t-BOOH 與 H2O2 抗致突變性較好之五株益生菌為材料，以 MTT 細胞毒性試驗檢測其抑制人類結腸癌細胞株 HT-29 及 Caco-2 增生及4-nitroquinoline-N-oxide (4NQO) 對腸道細胞 Int-407 細胞毒性之效果，並進一步利用分段式預反應探討其抗致突變的機制。此外，利用彗星電泳法 (comet assay) 檢測益生菌抑制4NQO誘導Int-407基因毒性之效果。結果顯示益生菌於高濃度 (108 CFU/mL) 時，抑制大腸癌細胞增生之效果較低濃度 (106 CFU/mL) 好。且隨著作用時間增加至48小時，抑制癌細胞增生之效果也更顯著，其中以108 CFU / mL 之 Bifidobacterium lactis Bb-12 的粗細胞壁抑制效果最佳，與 HT-29 及 Caco-2 作用 48 小時後之細胞存活率分別為 70.6 % 及 63.3 %。在抑制基因毒性方面，B. lactis Bb-12與L. casei 01 的熱致死細胞降低由 4NQO 所誘導的 Int-407 細胞 DNA 損傷情形最佳。在抗細胞毒性的機制方面，部分益生菌之粗細胞壁對 4NQO 具有去致突變 (desmutagenic effect) 的效果，其存活率可由 56.3 % 上升至 66.4 %；而益生菌之胞內液則具有阻斷作用 (blocking effect)，其細胞存活率可由 57.4 % 上升至 65.8 %。此外，除了益生菌菌體本身外，以 B. lactis Bb-12 及 L. casei 01 所發酵之牛乳上清液同樣可以抑制 4NQO 誘導 Int-407 之 DNA 損傷及細胞毒性，其抑制細胞毒性之機制同樣為去致突變及阻斷作用，其中去致突變之抑制率分別為 37.4 % 與 52.5 %，而阻斷作用之抑制率則分別為 34.1 % 與 41.4 %。

關鍵字

益生菌 ； 4-Nitroquinoline-N-oxide ； 彗星電泳 ； 去致突變 ； 阻斷作用

並列摘要

Colorectal cancer is a major cause of death from cancer in Taiwan. Approximately 70 % of colorectal cancer is related to dietary habit. Many researches showed that probiotics, both cellular compositions and fermented products have antimutagenic and antitumor activities on mutagens. In present study, we used several probiotics, which have been studied in our laboratory for their acid-resistance, bile salt tolerance, and antimutagenicity against B[a]P, t-BOOH, and H2O2. In addition, MTT assay was used to explore the effects of several probiotics against 4NQO cytotoxicity on Int-407 and the proliferation of human colon cancer cell HT-29 and Caco-2. Furthermore, possible antimutagenic mechanisms of probiotics were investigated by section-preincubation test and comet assay to explore the effects of several probiotics against 4NQO-induced DNA damage on Int-407. The results showed that a high probiotics population inhibits colon cancer cell more effectively than a low probiotics population. Among the strains, the crude cell walls of Bifidobacterium lactis Bb-12 exhibited the most potent inhibition activity in HT-29 and Caco-2 for 48h and the viability were 70.6 % and 63.3 %, respectively. The heat-killed cells of B. lactis Bb-12 and L. casei 01 significantly reduced the genotoxicity of 4NQO-induced DNA damage on Int-407 (p < 0.05). In addition, the crude cell walls and intracellular extracts of probiotics showed inhibitory effects against 4NQO cytotoxicity. The primary mechanism of crude cell walls was desmutagenic effect and intracellular extracts was blocking effect. Besides, fermented milk with B. lactis Bb-12 and L. casei 01 also had inhibition on 4NQO-induced DNA damage and cytotoxicity on Int-407. Their main mechanisms were the same as above, inhibition rate were 37.4 % - 52.5 % and 34.1 % - 41.4 %, respectively.

並列關鍵字

probiotics ； 4-Nitroquinoline-N-oxide ； comet assay ； desmutagenic effect ； blocking effect

參考文獻

行政院衛生署。2008。民國96年主要死因統計。行政院衛生署。台北。

許雅鈞。2004。雙叉桿菌胞內物對活性氧之抗致突變性與抗氧化性。國立台灣大學食品科技研究所碩士論文。台北。

陳惠英、顏國欽。1996。膳食中之抗致突變物及其作用機制。中華民國營養學會雜誌。21(3):323-38。

林國暉。2006。乳酸發酵蔬菜萃取物對4-nitroquinline-N-oxide基因毒性及數株細胞抑制之研究。國立台灣大學食品科技研究所碩士論文。台北。

洪瑞琳。2005。乳酸發酵蔬菜萃取物對4-Nitroquinoline N-oxide及Benzo[a]pyrene之抗致突變性硏究。國立台灣大學食品科技研究所碩士論文。台北。